In Trypanosoma brucei mitochondria, RNAs are synthesized polycistronically. Nevertheless, levels of mature monocistronic RNAs vary dramatically between life cycle stages. This indicates that steady-state RNA abundance, and thus gene expression, is controlled by posttranscriptional processes and these processes are developmentally regulated. Our hypothesis is that RNA decay pathways are critical determinants of gene regulation in this system. Our previous studies identified both cis- and trans-acting factors that define novel pathways of trypanosome gene regulation, and indicate that distinct decay pathways exist for unedited and edited mitochondrial RNAs. On unedited mRNA, the presence of a poly(A) tail significantly stimulates RNA turnover. In direct contrast, polyadenylation stabilizes edited RNAs. Moreover, the same small edited element that switches the poly(A) tail from a destabilizing to a stabilizing element is sufficient to facilitate rapid decay of edited RNA lacking a poly(A) tail compared to its unedited counterpart. These data reveal the presence of two exoribonuclease activities that we have shown are peripherally associated with mitochondrial membranes: (i) an activity that rapidly degrades poly(A+) unedited RNAs and (ii) an activity that specifically degrades edited RNAs that lack a poly(A) tail. Two candidate exoribonucleases were identified in the T. brucei genome, and one was shown to be essential for optimal growth. A third trans-acting enzyme involved in mitochondrial RNA turnover is the RET1 terminal uridylyltransferase (TUTase). In organello data implicate RET1 in facilitating turnover of a subset of poly(A+) RNAs, potentially through modification of their 3' tail sequences. The long-term goal of this project is to understand the roles of RNA turnover in trypanosome mitochondrial gene expression, and to elucidate the underlying biochemical mechanisms that regulate these events. The Specific Aims are: 1) Define cis-acting elements that regulate edited RNA stability. We will determine the range of edited RNAs that are stabilized by polyadenylation, and precisely define cis-acting stabilization sequences. We will use RNA structure determination and RNA-protein interaction assays to address the mechanism by edited cis-elements stabilize polyadenylated RNAs. 2) Identify and characterize exoribonucleases involved in mitochondrial RNA turnover. We will biochemically isolate the poly(A) RNA selective exoribonuclease and analyze its role in mitochondrial RNA metabolism using RNAi. Using a dual-RNAi strategy, we will identify the exoribonuclease that rapidly degrades non-adenylated edited RNAs. Here, candidate exoribonucleases will be ablated in cells engineered to produce increased levels of poly(A-) edited RNAs. 3) Determine the role of RET1 TUTase in mRNA 3' end formation and stability. Through analysis of RET1 knock-down cells, we will identify RNAs whose turnover is impacted by RET1 in vivo and assess their 3' end modifications. These studies will greatly increase our understanding of gene regulation in a medically and economically important parasite, and will provide important insights in to the mechanisms of mitochondrial gene regulation in higher organisms. PUBLIC HEALTH RELEVANCE: The long term goal of this project is to elucidate the roles of specific cis-acting elements and trans-acting factors in mitochondrial RNA decay in Trypanosoma brucei, and to understand the mechanisms by which these factors regulate mitochondrial gene expression. These studies will greatly increase our understanding of gene regulation in a medically and economically important parasite, and will provide important insights into the mechanisms of mitochondrial gene regulation in higher organisms. [unreadable] [unreadable] [unreadable]